Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies

Spatial reorganization of metabolic enzymes to form the “metabolic enzymes transiently assembling (META) body” is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27–42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed “META body-forming sequences (METAfos).” METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.

Introduction forming regions assume the possibility of multiple condensate-forming regions. The presence of condensate-forming amino acid regions in other META body-forming proteins is currently unknown. Further, the condensate-forming amino acid regions may be present in domains other than the N-terminus. Although the condensate-forming regions of Eno2p and Pfk2p appear to differ, further studies on other META body-forming proteins are required to determine the degree of variation among condensate-forming amino acid regions.
In the present study, we focused on pyruvate kinase (Cdc19p), a hypoxia-dependent condensate-forming enzyme of S. cerevisiae, and searched for condensate-forming regions by fragmenting the entire amino acid sequence. While the amino acid sequence of Cdc19p has not previously been studied with a focus on condensate formation under hypoxia, several amyloidforming regions of Cdc19p have been reported. Previous studies have reported that Cdc19p has a hydrophobic low-complexity region (LCR) at the C-terminus and aggregates under heat stress [16][17][18] in a phosphorylation-dependent manner [19] to form stress granules. Aggregation of Cdc19p is thought to inhibit enzyme activity, in contrast to the suggested function of condensates formed under hypoxia. In the present study, fragments of Cdc19p were formed to allow the detection of multiple condensate-forming regions, if present. The identified condensate-forming regions of Cdc19p and known condensate-forming regions of Eno2p were then used to test the effect of spatial reorganization of metabolic enzymes on cellular metabolism.

Validation of GFP strains
Integration of the GFP cassette in the genome of the S. cerevisiae CDC19-GFP and ENO2-GFP strains (Yeast GEP clone YAL038W, Thermo) [23] were confirmed by PCR amplification of the flanking region of CDC19 or ENO2 locus and Sanger sequencing (performed by Eurofins Genomics) using the primers listed in S1 Table in S1 File.

Yeast cell culture for overexpression of recombinant proteins
After transformation and culture on SDC+HLM agar plates, yeast colonies were picked and suspended in 100 μL of 4% (w/v) paraformaldehyde solution (Nacalai), fixed at 4ºC, and visualized using fluorescent microscopy (BZ-9000; Keyence, Osaka, Japan) equipped with a CFI Plan Apochromat Lamda x100 oil lens (NA = 1.45; Nikon Co., Tokyo, Japan) and a GFP-B filter (Excitation filter 470/40, Barrier filter 535/50, Dichroic mirror 500 nm; Nikon Co.). Images were obtained and analyzed using BZ-II Viewer Version 2.1.0 (Keyence) and BZ-II Analyzer Version 2.2 software. The foci-forming ratio of each transformant was calculated as described below with a total of 72-417 cells counted for each sample. Transformation of yeast cells was repeated three times for each plasmid on different dates to ensure reproducibility.

Calculation of the ratio of foci-forming cells
The ratio of foci-forming cells was calculated according to previously reported methods [3] using Katikati Counter Version 2.71 software (GTSOFT; https://www.vector.co.jp/soft/cmt/win95/art/ se347447.html). The ratio of foci-forming cells (%) was calculated as the number of foci-forming cells divided by the total number of cells.

Visualization of Cdc19p-derived peptide fragments using PyMOL
The PyMOL Molecular Graphics System (Version 2.5.2, Schrödinger, LLC) was used for visualization of Cdc19p peptide fragments. The three-dimensional structure of Cdc19p was downloaded from PDB (accession number 1A3W) [24].

Yeast cell culture for Cu 2+ -dose-dependent production of recombinant proteins under hypoxia
After culture of plasmid-transformed yeast in SDC+HLM agar medium, colonies were picked and pre-cultured in 5 mL of liquid SDC+HLM medium at 30˚C overnight in an incubator shaker at 300 rpm (RMS-25R-3; Sanki Seiki Co., Ltd., Osaka, Japan). Pre-cultured media was centrifuged at 2,300 ×g for 5 min at 4ºC. After discarding supernatants, cells were collected and inoculated into fresh SDC+HLM medium to achieve an OD 600 of 0.5. Suspended cells (1 mL) were transferred to 24-well plates with 10 μL of 0, 5, and 10 mM CuSO 4 stock solution added to make final concentrations of 0, 50, and 100 μM, respectively. For hypoxic culture, 24-well plates were placed in a multigas incubator (SD830, ASTEC Co., Ltd., Fukuoka, Japan) and statically incubated in 1.0% O 2 and 5.0% CO 2 for 24 h as previously described [10]. After hypoxic culture, 100 μL aliquots of culture media were used to measure OD 600 values. To calculate foci-forming ratios, 900 μL aliquots of culture media were centrifuged at 2,300 ×g for 5 min at 4ºC. After discarding supernatants, cells were washed with 1 mL of 1× PBS (pH 7.4) and centrifuged 2,300 ×g for 5 min at 4ºC. After discarding supernatants, cells were resuspended in 500 μL 4% (w/v) paraformaldehyde solution, fixed at 4ºC, and visualized using fluorescent microscopy. Foci-forming ratios were calculated as described above with a total of 110-357 cells counted for each sample. The transformation of yeast cells with each plasmid followed by cell culture was repeated three times on different dates to ensure reproducibility.

Preparation of cells for colocalization analysis
CDC19-GFP or ENO2-GFP strains were transformed with the CUP1 promoter-dependent ADH1-expressing plasmids and cultured on SDC+HLM agar media. Colonies were inoculated in 5 mL SDC+HLM medium and cultured in an incubator shaker at 300 rpm for 24 h at 30ºC. Cells were inoculated into fresh SDC+HLM medium to achieve an OD 600 of 0.05 then 10 mM of CuSO 4 stock solution was added to a final concentration of 100 μM. After pre-culture in a shaker incubator at 300 rpm for 24 h at 30ºC, culture medium was inoculated into fresh SDC +HLM medium to achieve an OD 600 of 0.05 then 10 mM of CuSO 4 stock solution was added to a final concentration to 100 μM. Cell suspensions were transferred to 24-well plates in 1 mL aliquots and cultured in a multigas incubator with 1.0% O 2 and 5.0% CO 2 for 6 h (ENO2-GFP strains) or 24 h (CDC19-GFP strains). After hypoxic culture, culture media was transferred to a 1.5 mL tube and centrifuged at 2,300 ×g for 5 min at 4ºC. After discarding supernatants, cells were washed with 1 mL of 1× PBS (pH 7.4) and centrifuged 2,300 ×g for 5 min at 4ºC. After discarding the supernatants, the cells were resuspended in a 500 μL solution of 4% (w/v) paraformaldehyde, fixed at 4ºC, and visualized using fluorescent microscopy. Colocalization ratios were calculated using a total of 30-179 cells with both green and red foci for each sample. The transformation of yeast cells with each plasmid, followed by cell culture, was repeated three times on different dates to ensure reproducibility.

Construction of an ADH1 knockout strain
Primers and plasmids used for construction of the ADH1 knockout strain are listed in S1 and S2 Tables in S1 File, respectively. The KanMX6 fragment was amplified from the pFA6a-GFP (S65T)-kanMX6 plasmid. S. cerevisiae BY4741 competent cells were transformed with the amplified KanMX6 fragment using yeast transformation kit. Transformants were plated on YPD agar plates containing 600 ng/mL G418 and cultured at 30ºC for one week. Obtained colonies were streaked on YPD agar plates containing 600 ng/mL G418 twice and colonies were checked by colony PCR. Obtained colonies were cultured in 5 mL of YPD medium containing 200 ng/mL G418. After extraction of DNA, flanking regions of ADH1 locus were amplified by PCR and knockout of ADH1 was confirmed by Sanger sequencing (performed by Eurofins Genomics). The constructed adh1Δ strain was deposited in the National Bio-Resource Project (NBRP), Japan (http://www.nbrp.jp/).

Measurement of growth rate of yeast strains
BY4741 wt and adh1Δ strains were transformed with the ADH1-overexpression plasmid pULI-G2-ADH1 and cultured on SDC+HLM agar media. Uracil auxotrophic colonies were inoculated into 5 mL of SDC+HLM media and incubated in a shaking incubator at 300 rpm for 24 h at 30˚C. Pregrown yeast cells were then inoculated into three tubes containing 5 mL of fresh SDC+HLM medium to achieve an OD 660 of 0.05. OD 660 was measured prior to incubation (time 0), and then at 1, 3, 6, 9, 12, 24, 30, and 48 h after starting incubation using a Taitec Mini-Photo 518R spectrometer (TAITEC Coporation, Saitama, Japan). Three independently isolated transformants were used to ensure reproducibility. For comparison, the growth rate of plasmid-less BY4741 wt and adh1Δ were also measured using YPD and SDC+HLMU media as described above. The experiment was repeated three times. Growth rate was calculated as the "increase in OD 660 " using following formula: increase in OD 660 = OD 660 (at each time point) -OD 660 (time 0).

Preparation of cells for metabolite analysis
ADH1-knockout cells were transformed with plasmids and cultured in SDC+HLM agar media. Colonies were inoculated in 5 mL SDC+HLM medium and cultured in an incubator shaker at 300 rpm for 24 h at 30ºC. Cells were inoculated into fresh SDC+HLM medium to achieve an OD 600 of 0.05 then 10 mM of CuSO 4 stock solution was added to a final concentration of 100 μM. For this experiment, a portable reader PiCOEXPLORER (PAS-110-YU; Yamato Scientific Co., Ltd., Tokyo, Japan) was used to measure OD 600 . After pre-culture in a shaker incubator at 300 rpm for 24 h at 30ºC, culture medium was inoculated into fresh SDC +HLM medium to achieve an OD 600 of 0.05 then 10 mM of CuSO 4 stock solution was added to a final concentration to 100 μM. Cell suspensions were transferred to 24-well plates in 1 mL aliquots and cultured in a multigas incubator with 1.0% O 2 and 5.0% CO 2 for 6 h. After hypoxic culture, cells were collected by centrifuging at 500 × g for 5 min at 4ºC. Supernatants were discarded and pelleted cells were resuspended in fresh SDC+HLM medium without glucose and washed by centrifuging at 500 ×g for 5 min at 4ºC. After discarding supernatants, cells were suspended in fresh SDC+HLM medium without glucose to achieve an OD 600 of 0.1. Cell suspensions were transferred to 96-well plates in 50 μL aliquots and fresh SDC+HLM medium containing 4% (w/v) glucose was added to a total volume of 100 μL. Cells were then statically cultured at 30ºC using a plate incubator (FRONT LAB; AS ONE Co., Osaka, Japan). After incubation for 0, 2, 10, 30, 60, 90, and 120 min, 100 μL of 10 mM H 2 SO 4 was added to each well. After measuring OD 600 values using the PiCOEXPLORER reader, cell suspensions were transferred to 0.2 mL tubes and centrifuged at 20,400 ×g for 10 min at 4ºC. Supernatants were used for HPLC analyses.

HPLC analyses
The glucose, ethanol, acetic acid, and glycerol contents of samples were measured using an HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a refractive index detector (RID-10A, Shimadzu) and an Aminex HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 40ºC as previously described [25]. As a mobile phase, 5 mM H 2 SO 4 was used at a flow rate of 0.6 mL/min. The concentrations of each metabolite were calculated using analysis software (LCsolution Version 1.25 SP4, Shimadzu) based on calibration curves prepared using standards for each substance.

Measurement of cell fluorescence intensity
BY4741 or ADH1-knockout cells transformed with plasmids were cultured in hypoxic conditions as described above. Each strain was cultured in three wells of a 24-well plate in all experiment.

Yeast protein extraction and Western blotting analysis
BY4741 or ADH1-knockout cells transformed with plasmids were cultured in hypoxic conditions as described above. Each strain was cultured in eight wells of a 24-well plate in all experiments. After 6 h, yeast cells were collected by centrifuging at 3000 × g for 10 min at 4ºC. Supernatants were discarded and pelleted cells were washed in sterile water. Yeast cells were pelleted again and stored at -80ºC until use. Yeast cells were lysed using Y-PER™ Yeast protein extraction reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Total yeast lysates were centrifuged at 500 × g for 5 min at 4 ºC, and the supernatants were transferred to new tubes to obtain soluble proteins. The protein concentration of samples was determined by using a protein assay BCA kit (Nacalai Tesque). Soluble proteins (10 μg and 20 μg) for BY4741 and adh1Δ transformants, respectively, were separated by SDS-PAGE with 5-20% e-PAGEL (ATTO, Tokyo, Japan), and then transferred onto nitrocellulose membranes (Bio-Rad, Tokyo, Japan), using a semidry blotting apparatus HorizeBLOT (ATTO). A Western blot analysis was performed using a polyclonal rabbit anti-ADH (yeast) polyclonal antibody conjugated with horseradish peroxidase (HRP) (Rockland Immunochemicals Inc., PA, USA). As a loading control, β-actin was detected using a polyclonal rabbit-anti-β-actin primary antibody (GenTex Inc., CA, USA) (1:5000 dilution) and a polyclonal goat-anti rabbit IgG (H+L) secondary antibody conjugated with HRP (Proteintech Group, Inc., IL, USA) (1:10000 dilution). The chemiluminescence was detected using Chemi-Lumi One Super and Chemi-Lumi One Ultra (NacalaiTesque) for Adh1p and β-actin, respectively. The signals on the membranes were captured by using the luminescent imaging analyzer LAS-4000 (Fujifilm, Tokyo, Japan). The band intensity for Adh1p and β-actin were quantified using ImageJ software (National Institute of Health (NIH), USA). The relative amount of Adh1p was calculated as (band intensities by anti-ADH1 treatment)/(band intensities by anti-β-actin).

Statistical analyses
All experiments were independently repeated three times. Unless otherwise noted, the F-test and Student's t-test were used to evaluate differences between groups. P-values <0.05 were considered statistically significant.

Identification of Cdc19p-derived peptides that form foci in S. cerevisiae when conjugated with fluorescent proteins
To determine whether Cdc19p forms foci as reported for Eno2p and Pfk2p, 33 fragments of Cdc19p conjugated with EGFP were prepared. Fig 1A shows the foci-forming ratio of Cdc19pderived peptide fragments conjugated with EGFP and overexpressed in S. cerevisiae using plasmids under normoxia, as previously reported [3]. The fragmentation pattern of Cdc19p ( Fig  1B) was determined with consideration of the two-dimensional structure of Cdc19p. Most peptide fragments formed foci; however, fragments from the N-terminal region (1-32 a.a.) and dimer formation interface (258-372 a.a.) did not. Peptide fragments that formed foci were further shortened in a stepwise manner to obtain peptide fragments approximately 20-40 a.a. in length. Four representative peptide fragments; SC1 (33-74 a.a.), SC2 (129-158 a.a.), SC3 (217-243 a.a.), and SC4 (373-404 a.a.), were located at the surface of Cdc19p (Fig 2A), indicating that these regions may contribute to intermolecular interactions. Fig 2B shows representative images of foci formed by each EGFP-conjugated fragment under normoxia. EGFP (produced by plasmid pUL-ATG-EGFP) and full-length Cdc19p fused with EGFP (produced by plasmid pULGI2-CDC19) did not form condensates, while SC1, SC2, SC3, and SC4 fragments fused with EGFP (produced by plasmids pULGI2-SC1, -SC2, -SC3, and -SC4, respectively) formed condensates under normoxia. SC2 and SC3 were selected for use in further experiments as they were shorter with higher foci-forming ratios compared to SC1 and SC4.

Effect of Adh1p incorporation into META body on cell metabolism
To test the effect of artificial enzyme assemblies on cell metabolism, peptide or protein fragment-tagged Adh1p-FusionRed proteins were produced in ADH1-knockout cells under hypoxia (Fig 4A). In this experiment, the proliferation of ADH1-knockout cells was reduced (S4 Fig in S1 File) and some plasmid transformants were not obtained; therefore, some plasmids were not used in this experiment. The growth defect of ADH1-knockout cells was recovered by the introduction of plasmids for overexpressing ADH1 (S4 Fig in S1 File), suggesting that the growth defect was due to the non-existence of ADH1. Most enzymes of the glycolytic pathway that catalyze the metabolism of glucose to acetaldehyde and acetaldehyde to acetic acid are known to form condensates under hypoxia, while enzymes involved in glycerol and ethanol synthesis do not [9] (4B Fig). If the localization of Adh1p to intracellular META bodies or other condensates has no effect on cell metabolism, then the production of ethanol from glucose and the ratio of ethanol to acetic acid in culture media should be unchanged.
The foci-forming ratios of Adh1p-FusionRed and peptides or domains conjugated with Adh1p-FusionRed in ADH1-knockout cells are shown in Fig 4C. While Adh1p-FusionRed formed a small number of foci, SC3, scENO, FUSN, and Sup35p-conjugated Adh1p-FusionRed had significantly higher foci-forming ratios compared to Adh1p-FusionRed. The foci-forming ratios of cells producing SC3-or scENO-conjugated Adh1p-FusionRed were approximately 10%, while that of FUSN-or Sup35p-conjugated Adh1p-FusionRed were higher than 40%. These results indicate that, to varying degrees, artificial assembly of Adh1p was induced by the transformants. Cellular fluorescence per cell density was similar between   was significantly higher in scENO-conjugated Adh1p-FusionRed-producing cells compared to Adh1p-FusionRed-producing cells, indicating that cellular metabolism was altered in response to changes in spatial localization of Adh1p following conjugation with scENO. The concentrations of acetic acid (S7B Fig in S1 File), glycerol (S7C Fig in S1 File), and glucose (S7D Fig in S1 File) in the culture media of scENO-conjugated Adh1p-FusionRed-producing cells were similar to that of Adh1p-FusionRed-producing cells.

Identification of foci-forming regions in Cdc19p
In the present study, fragmentation of Cdc19p identified four peptide fragments, namely SC1, SC2, SC3, and SC4, as foci-forming regions. The SC4 region (373-404 a.a.) overlaps the previously reported amyloid-forming LCR region (KPTSTTETVAASAVAAVFEQK, 374-394 a.a.; Table 2 and S3 Table in S1 File) [16,19], indicating that the method used in the present study can also be applied to the identification of amyloid-forming regions. Cdc19p is known to have two catalytic domains at 19-88 a.a. and 189-360 a.a., a capping domain at 89-188 a.a., and a regulatory domain at 361-500 a.a. [24]. The SC1 and SC3 regions are located within catalytic domains, SC2 is located in the capping domain, and SC4 is located in the regulatory domain of Cdc19p. Previously reported post-translational modification sites in Cdc19p were not found in SC1, while SC2, SC3, and SC4 contained 2, 4, and 8 sites, respectively. No obvious similarities were observed between the SC1, SC2, SC3, and SC4 regions in terms of two-dimensional structure, amino acid composition, hydrophobicity, or estimated water solubility (S3 Table in S1 File). Accordingly, further studies are required to determine the contributions of the SC1, SC2, SC3, and SC4 regions to the formation of Cdc19p condensates under hypoxic conditions. We believe this to be the first report of multiple focus-forming regions within a single protein as the previously reported condensate-forming enzymes, Eno2p and Pfk2p, have a single focus-forming region.
The location of the SC1, SC2, SC3, and SC4 regions of Cdc19p on the molecular surface (Fig 2A) suggests that these regions are involved in molecular interactions within Cdc19p or between Cdc19p and other cellular components. Conjugation of the SC2 and SC3 regions with Adh1p and FusionRed resulted in the co-localization of these fusion proteins with META bodies formed by Cdc19p and Eno2p under hypoxia (Fig 3B and S3 Fig in S1 File), indicating that these regions may be involved in the formation of Cdc19p condensates under hypoxia. Investigating whether these regions interact with other cellular components or form condensates in vitro would contribute to understanding of the molecular mechanisms of intracellular foci formation. The contribution of these regions to the condensate formation by Cdc19p should also be investigated. The formation of foci by SC fragments fused to Adh1 has been observed in both normoxic and hypoxic conditions (S1 and S3 Figs in S1 File, respectively). Testing the existence of foci formed by Cdc19p fragments in cells with eliminated META bodies would be useful in determining whether the SC fragments form foci in a META body-dependent manner.
The mechanisms regulating the formation of condensates by glycolytic enzymes are not completely known yet, and it has been reported that certain gene knockouts [3,4] or chemical treatment [3,10] can inhibit condensate formation. However, it is currently difficult to completely inhibit condensate formation through gene knockout or chemical treatment without affecting cell proliferation. Previous studies have attempted to identify conclusive regulators of glycolytic enzyme condensate formation, but the results suggest that each enzyme may have its own regulatory machinery and corresponding amino acid residues, meaning that there may not be a single, conclusive regulator for the formation of these condensates. These results may contribute to a better understanding of the regulatory machinery behind the formation of glycolytic enzyme condensates.

Artificial intracellular enzyme assembly of Adh1p
The conjugation of Adh1p with foci-forming regions (SC2, SC3, and scENO), IDR (FUSN), and amyloid-forming region (Sup35p) resulted in artificial intracellular enzyme assembly ( Fig  3B). As expected, the enzyme assemblies formed by SC2, SC3, and scENO colocalized with META bodies while enzyme assemblies formed by FUSN and Sup35p had different localizations indicating that the molecular mechanisms underlying the formation of META bodies differs to that of FUSN and Sup35p.
During protein expression by the CUP1 promoter, the foci-forming ratio of Adh1p conjugated with FusionRed and peptide fragments increased in a Cu 2+ -dependent manner for SC2, SC3, scENO, and FUSN while the production of Sup35p was not Cu 2+ dependent (Fig 3C) with protein production observed even without the addition of Cu 2+ to the culture medium. These findings indicate amyloid formation induced by Sup35p is not largely dependent on intracellular protein concentration.
The foci-forming ratio of SC3 conjugated with Adh1p and FusionRed was approximately four times lower for CUP1 promoter-dependent expression compared to GAPDH promoterdependent expression (S1 Fig in S1 File and Fig 4C) indicating that the intracellular protein level is a major determinant of the foci-forming ratio. Unexpectedly, Adh1p conjugated with EGFP also formed foci when overexpressed under the GAPDH promoter in wild type cells (S1 Fig in S1 File) suggesting that Adh1p can form intracellular condensates. While yeast Adh1p has been posited to form soluble aggregates under heat treatment dependent on the amino acid residues 40-60 [28], the ability of Adh1p to form condensates under growth conditions has not previously been studied. As commercially available GFP clone collections [23] do not currently contain ADH1-GFP strains, the construction of ADH1-GFP strains and studies of the formation of Adh1p condensates under hypoxia are required. Despite this, the finding that the ratio of assembly-forming cells can be altered using peptide fragments (Fig 4C) with normal or decreased intracellular Adh1p levels ( S5 Fig in S1 File) demonstrates the effects of artificial enzyme assemblies formed by Adh1p can be studied in vivo. However, the limitations of using Adh1p, including the slow growth of the ADH1 knockout strain and the formation of foci when overexpressing Adh1p, should be carefully considered. In the present study, SC2, SC3, and scENO, that can localize proteins to META bodies in vivo, were named as "META body-forming peptide sequence tags (METAfos-tag)."

Effects of intracellular artificial enzyme assembly on cellular metabolites
Of the METAfos-tags, yeast cells producing scENO-tagged Adh1p-FusionRed had increased ethanol per acetic acid concentration, indicating that the localization of Adh1p to META bodies affects cell metabolism, although this change was subtle. This finding may be attributable to Adh1p-FusionRed alone being able to form foci, thereby confounding the effect of scENOtagged Adh1p-FusionRed on foci formation. Further, the expression of ADH5, a paralogue of ADH1 with similar function, may confound the results of the present study; however, this effect may be minimal as the intracellular protein concentration of Adh5p is reported to be approximately 19 times lower than Adh1p (Saccharomyces Genome Database, https://www. yeastgenome.org/). To test the effect of spatial reorganization of metabolic enzymes on cellular metabolism, novel methods are needed to increase foci formation using peptide tags that reduce the impact on protein production in addition to recruiting other proteins.
To conclude, the results of the present study demonstrate that the fragmentation of a META body-forming protein enables the identification of foci-forming peptides of approximately 20-40 a.a. in length. We further demonstrated that conjugating foci-forming peptides allows the co-localization of proteins with META bodies under hypoxia and the formation of molecular condensates that are distinct from those formed by representative liquid-liquid phase separating peptide fragments or amyloid-forming peptide fragments. Further elucidating the mechanisms underlying the effects of this region on foci formation may facilitate the development of strategies for regulating cellular metabolism through the spatial reorganization of metabolic enzymes.